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The study, published in Science, describes an approach that efficiently forms single-copy HACs, bypassing a common hurdle that has hindered progress in this field for decades.
Artificial chromosomes are lab-made structures designed to mimic the function of natural chromosomes, the packaged bundles of DNA found in the cells of humans and other organisms. These synthetic constructs have the potential to serve as vehicles for delivering therapeutic genes or as tools for studying chromosome biology. However, previous attempts to create HACs have been plagued by a major issue: the DNA segments used to build them often link together in unpredictable ways, forming long, tangled chains with rearranged sequences.
The Penn Medicine team, led by Dr. Ben Black, sought to overcome this challenge by completely overhauling the approach to HAC design and delivery. "The HAC we built is very attractive for eventual deployment in biotechnology applications, for instance, where large-scale genetic engineering of cells is desired," Dr. Black explains in a media release. "A bonus is that they exist alongside natural chromosomes without having to alter the natural chromosomes in the cell."
To test their idea, the scientists turned to a tried-and-true workhorse of molecular biology: yeast. They used a technique called transformation-associated recombination (TAR) cloning to assemble a whopping 750 kilobase DNA construct in yeast cells. For context, that's about 25 times larger than the constructs used in previous HAC studies. The construct contained DNA from both human and bacterial sources, as well as sequences to help seed the formation of the centromere.
The next challenge was to deliver this hefty payload into human cells. The team accomplished this by fusing the engineered yeast cells with a human cell line, a process that had been optimized in previous studies. Remarkably, this fusion approach proved to be much more efficient than the traditional method of directly transferring naked DNA into cells.